一种简单有效的基于阳离子交换层析的重组腺相关病毒纯化方法  

A Simple and Effective Purification Method of Recombinant Adeno-associated Virus Based on Cation Exchange Chromatography

作　　者：周豪杰 王晨旭[1] 马强[2] 李媛[2] 高传玉[1] 张守涛[2] ZHOU Haojie;WANG Chenxu;MA Qiang;LI Yuan;GAO Chuanyu;ZHANG Shoutao(Peopl's Hospital Affiliated to Zhengzhou University,Zhengzhou,450000,China;College of Life Sciences,Zhengzhou University,Zhengzhou,450000,China)

机构地区：[1]郑州大学附属人民医院,郑州450000 [2]郑州大学生命科学院,郑州450000

出　　处：《病毒学报》2021年第3期605-612,共8页Chinese Journal of Virology

基　　金：国家自然科学基金(项目号:U1604184),题目:VEGF165影响心肌细胞膜L型钙通道电流在缺血再灌注中的机制研究。

摘　　要：本研究旨在探索出一套简单有效的重组腺相关病毒(rAAV)纯化方法,为基因治疗提供有力载体,推进rAAV的基因治疗走进临床。我们选用国际通用的无辅助病毒包装体系,将pAAV-cTnT-eGFP,pAAV-R2C9和pHelper三种质粒共转染HEK 293T细胞系进行病毒包装,并通过荧光显微镜观察包装效率;应用阳离子交换层析法对收获处理后的病毒液进行纯化,包括第Ⅰ轮高pH下的除杂纯化和第Ⅱ轮以严格密集梯度洗脱为特点的进一步纯化。通过SDS-PAGE和Western Blot(WB)对纯化产物进行检测,使用Q-PCR法测定病毒滴度,最后再通过透射电镜对病毒颗粒进一步验证和分析。荧光显微镜结果显示,病毒包装效率较高,可达70%~80%;SDS-PAGE结果表明病毒纯化效果显著,第Ⅰ轮纯化除去了大量的蛋白杂质,第Ⅱ轮纯化也明显除去部分杂质;WB和电镜结果证明,纯化产物实为rAAV病毒,经Q-PCR测得病毒的滴度可达2.15×10^(12)GC/mL,而且电镜进一步观察到我们纯化的终成品病毒中空颗粒含量极少,包含基因的实心颗粒占比可达99%。本研究成功开发出了一套的简单方便、切实有效的rAAV纯化方法,并且该方法能有效去除空壳病毒,提高包含基因的实心病毒占比。This study is to explore a simple and effective method for the purification of recombinant adeno-associated virus(rAAV),to provide a powerful vector for gene therapy,to promote rAAV gene therapy into the clinic.We used the international‘helpler-free’AAV production protocol and transfected the three plasmids pAAV-cTnT-eGFP,pAAV-R2 C9,and pHelper into HEK 293 T cell line with PEI transfection reagents.We observed the packaging efficiency by fluorescence microscope.The cation exchange chromatography was used to purify the virus solution after harvesting,including the first round of high-pH depuration purification and the second round of further purification characterized by strict intensive gradient elution.The purified product was detected by SDS-PAGE and Western blot(WB),and the virus titer was determined by Q-PCR method.Finally,the virus particles were further verified and analyzed by transmission electron microscopy.The results of fluorescence microscopy showed that the packaging efficiency of the virus was high,up to 70%~80%;SDSPAGE results showed that the virus purification effect was significant.The first round of purification removed a lot of protein impurities,and the second round of purification also significantly removed some impurities.The purified product was confirmed to be rAAV virus by WB and electron microscopy.The titer of the virus was2.15×10^(12) GC/mL measured by Q-PCR method,and the electron microscopy results further revealed that the empty particles contained in the final virus production were very few,and the percentage of genome containing vectors can reach 99%.A simple,convenient and effective method for the purification of rAAV was successfully developed in this study.And this method can also effectively remove the empty particles to increase the proportion of genome containing viruses.

关 键 词：重组腺相关病毒 离子交换层析 rAAV纯化 

分 类 号：R541.4[医药卫生—心血管疾病] R373.9[医药卫生—内科学]